Preparation of factor XIIIa by gene manipulation

ABSTRACT

The cDNA which codes for factor XIIIa has been isolated using a cDNA bank from human placenta and probes constructed on the basis of the amino acid sequence of factor XIIIa peptide fragments. It is possible with this cDNA not only to obtain factor XIIIa by gene manipulation in high purity but also to prepare diagnostic aids which permit the analysis of genetic factor XIIIa defects. Furthermore, it is possible on the basis of the amino acid sequence to prepare antibodies which are suitable for diagnostic aids and antibody columns.

This is a continuation of application Ser. No. 08/032,171, filed Mar.12, 1993, which is a continuation application of Ser. No. 07/549,234,filed Jul. 9, 1990, abandoned, which is a continuation application ofSer. No. 07/024,174 filed Mar. 10, 1987, abandoned.

Coagulation factor XIII is the final member of the “coagulation cascade”in the natural process of blood coagulation in vertebrates. Theenzymatically active form of factor XIII, factor XIIIa, also called“activated fibrin-stabilizing factor”, “fibrinoligase” or “plasmatransglutaminase” and, hereinafter, “F XIIIa”, catalyzes the fusion offibrin units in preexistent thrombi by intramolecular crosslinking(Lorand et al., Methods in Enzymology 80 (1981), 333-341; Curtis et al.,Annals New York Academy of Sciences 1983, 567-576). The molecular weightof factor XIII from plasma is about 300 kD (Loewy et al., J. Biol. Chem.236 (1961) 2634). The molecular weight of the active subunit F XIIIa isabout 80 kD (Bohn and Schwick, Arzneimittelforschung 21 (1971) 1432).During the activation of factor XIII, thrombin splits off from theprecursor a peptide which is about 4 kD in size and has a known sequenceof 36 amino acids (Takagi and Doolittle, Biochemistry 13 (1974)750-756). In addition, a sequence embracing four amino acids is known(Holbrook et al., Biochem. J. 135 (1973) 901-903).

The invention relates to a process for the preparation of F XIIIa bygene manipulation, to the mRNA necessary for this, to the cDNA obtainedtherefrom, to DNA structures and vectors containing all or part of thiscDNA, to cells transformed with DNA of this type, and to the polypeptideexpressed by these cells. The invention also relates to part-sequencesof the amino acid sequence of F XIIIa, to specific antibodies obtainedtherewith, to diagnostic aids and antibody columns produced from theseantibodies, and to a polypeptide obtained with the aid of such columns.Another aspect of the invention relates to diagnostic aids which containall or part of the DNA or RNA coding for F XIIIa, and to diagnosticmethods with which body fluids and tissues are examined using diagnosticaids of this type. Further aspects of the invention and its preferredembodiments are illustrated in detail hereinafter and defined in thepatent claims.

The drawings, in which the numbers coincide with those in the examples,illustrate the invention:

FIG. 1 shows the cDNA coding for F XIIIa (the coding region beingshaded) and, below this, the DNA regions of the isolated andcharacterized clones.

FIG. 2 shows the construction of the expression plasmid pFXIII-13. Forclarity, in this figure the starting plasmids pIC19H-12.1 andpIC19H-11.1, as well as the DNA fragments located immediately belowthem, are represented by double lines, as is the product pFXIII-13constructed from the single-stranded fragments.

FIG. 3 is a diagram of the construction of the plasmid pTrc97A, FIG. 3 athat of pFXIII-C4 from pTrc97A and pFXIII-13, and finally FIG. 3 b theconstruction of pMB259 from pFXIII-13 and the known plasmids pIC20H andpBD2.

FIG. 4 is a diagram of the construction of the plasmid pMB240 frompFXIII-13 and the known plasmid pAAH5.

FIG. 5 shows the construction of pZET4 from the known plasmid pSV2dhfrand the plasmid pSVA STOP1, FIG. 5 a shows the construction of pSVF13from pSVA STOP1 and pFXIII-13, FIG. 5 b shows the construction of pZF13from pZET4 and pFXIII-13, and finally FIG. 5 c shows the construction ofpHSF13 from pSVF13 and the known plasmid pSP6HS9.

FIG. 6 discloses the amino acid sequence of Factor XIIIa.

The amino acid sequence of F XIIIa fragments was determined for theconstruction of suitable probes. The corresponding peptide fragmentswere obtained by proteolysis or cleavage with cyanogen bromide. Based onknowledge of the amino acid sequences of such fragments, twooligonucleotide probes were synthesized, one 20mer and one 66mer.

In the 20mer probe all theoretically possible codons for the amino acidsequence

Met-Met-Asp-Ile-Thr-Asp-Thr

were taken into account, with, in the case of the last amino acid, thethird position in the codon being omitted. The 20mer probe is thus48-fold degenerate, i.e. a mixture of all 48 theoretically possibleoligonucleotides coding for the said amino acid sequence (Table 1;Appendix).

The 66mer probe was selected on the basis of the following amino acidsequence

Tyr-Gly-Gln-Phe-Glu-Asp-Gly-Ile-Leu-Asp-Thr-Cys-Leu-Tyr-Val-Met-Asp-Arg-Ala-Gln-Met-Asp

and with the assistance of statistical data (Lathe, J. Molec. Biol. 183(1985) 1-12) (Table 2, Appendix).

These probes were used to screen a cDNA bank. The cDNA was prepared frommRNA from a mature human placenta, the mRNA being isolated from thelatter, and the cDNA being prepared therefrom. The cDNA was providedwith EcoRI ends and ligated into the EcoRI cleavage site of the phagevector λgt10. A positive clone, λgt10-12, which was identified with theabovementioned probe, was further analyzed (FIG. 1). The sequencing, bymethods known per se, resulted in the DNA sequence which codes for FXIIIa.

Rescreening of the cDNA bank with this DNA sequence resulted inisolation of further clones which expand both towards the 5′- andtowards the 3′-end.

FIG. 1 shows the restriction map of the DNA sequence which codes for FXIIIa. “N” designates the N-terminal end and “C” designates theC-terminal end of the coding region, and “A₍₈₉₎” designates the poly(A)sequence of 89 bases. This sequence represents the whole of the codingsequence of F XIIIa. Table 3 (Appendix) shows the DNA sequence found(coding strand) and, deduced therefrom, the amino acid sequence from thecloned cDNA fragments from λgt10-11 and λgt10-12. The total length ofthe cDNA is 3905 base-pairs. The N-terminal sequence embracing 36 aminoacids found by Takagi and Doolittle (loc. cit.) is present in thesequence which was found. This sequence is indicated in Table 3 with anunbroken line between nucleotide positions 88 and 198. In addition tothe sequence found by Takagi and Doolittle, the cDNA codes for a valinein nucleotide positions 187-189. The sequence embracing four amino acidsfound by Holbrook et al. (loc. cit.)—Gly-Gln-Cys-Trp—is coded for by thecDNA in positions 1021-1032. This sequence is likewise indicated by anunbroken line. In addition, the positions of the 20mer and 66meroligonucleotide probes are indicated by broken lines. The 20mer probehybridizes between positions 1507 and 1526, and the 66mer probehybridizes between positions 766 and 831.

It is possible according to the invention to use the coding cDNA for thepreparation of modified genes which code for proteins having alteredbiological properties. It is possible for this purpose to undertake, ina manner known per se, deletions, insertions and base-exchanges.

It is also possible, by the choice of the host, to influence the natureof the modification to the F XIIIa. Thus, there is no glycosylation inbacteria, while that taking place in yeast cells differs from that inhigher eukaryotic cells.

Knowing the amino acid sequence of F XIIIa, it is possible to prepare,by conventional methods or gene manipulation, part-sequences of aminoacids which can act as antigens for the preparation of polyclonal ormonoclonal antibodies. Such antibodies can be used not only fordiagnostic purposes but also for the preparation of antibody columnswith which it is possible to remove F XIIIa from solutions which containthis factor in addition to other proteins.

It is also possible, by use of the cDNA or parts thereof,straightforwardly to isolate from a genomic bank the genomic clone whichcodes for F XIIIa and using which it is possible not only to express itin eukaryotic cells but also to gain further diagnostic information.

F XIIIa deficiencies can result in various syndromes which, to a largeextent, are attributed to the inability to convert the precursors intothe active form of the enzyme. Knowledge of the cDNA of F XIIIa nowpermits the preparation of diagnostic aids with which it is possiblestraightforwardly to establish whether genetic modifications arepresent.

Thus, it is possible according to the invention to prepare a highly purefactor XIIIa without any risk of contamination by, for example, virusesor other proteins. The dependence, which has existed to date, on humanplasma or placentae as source of raw material has thus been overcome. Inaddition, the invention allows access to valuable diagnostic aids andthus the analysis of genetic F XIIIa defects.

The invention is illustrated in detail in the examples which follows.Unless otherwise stated, percentages relate to weight where they do notrelate to amounts.

Apart from those explained in the text, the following abbreviations havebeen used:

EDTA = sodium ethylenediaminetetraacetate SDS = sodium dodecyl sulfateDTT = dithiothreitol BSA = bovine serum albumin

EXAMPLES

1. Isolation of RNA from human placenta

RNA was obtained from a mature human placenta (by the method of Chirgwinet al., Biochemistry 18 (1979) 5294-5299). About 10 g of placentaltissue was ground in liquid nitrogen, suspended in 80 ml of 4 Mguanidinium thiocyanate containing 0.1 M mercaptoethanol and treated ina homogenizer (Ultraturrax) at 20,000 rpm for 90 sec. The lysate wascentrifuged at 7,000 rpm for 15 min. (Sorvall GSA rotor) and 2 ml of 1 Macetic acid and 60 ml of abs. ethanol were added to the supernatant,which was allowed to precipitate at −20° C. overnight. Aftersedimentation at 6,000 rpm and −10° C. for 10 min, the nucleic acidswere completely dissolved in 40 ml of 7.5 M guanidinium hydrochloride(pH 7.0) and precipitated with a mixture of 1 ml of 1 M acetic acid and20 ml of abs. ethanol. To remove the DNA the precipitation was repeatedonce more with half the volumes. The RNA was dissolved in 12 ml of H₂O,precipitated with a mixture of 1.2 ml of 4 M potassium acetate and 24 mlof abs. ethanol, sedimented and finally redissolved in 10 ml of H₂O (1ml per g tissue).

Isolation of placental mRNA containing poly(A)

To isolate mRNA containing poly(A), the placental RNA was fractionatedby oligo(dT)-cellulose chromatography (Aviv and Leder, Proc. Natl. Acad.Sci. USA 69 (1973) 1408-1412) in 2 ml Pasteur pipettes in LiCl. About 5mg of placental RNA were applied to the column in buffer 1 (500 mM LiCl,20 mM tris (pH 7.5), 1 mM EDTA, 0.1% SDS). Whereas the poly(A)⁺ RNA wasbound to the oligo(dT)-cellulose, the poly(A)⁻ RNA could be elutedagain. After a wash with buffer 2 (100 mM LiCl, 29 mM tris (pH 7.5), 1mM EDTA, 0.1% SDS), the poly(A)⁺ RNA (placental mRNA) was eluted fromthe column with buffer 3 (5 mM tris (pH 7.5), 1 mM EDTA, 0.05% SDS).

For further purification, the poly(A)⁺ RNA was adjusted to buffer 1 andrechromatographed on oligo(dT)-cellulose. After this second purificationstep, the yield of placental poly(A)⁺ RNA was about 4% of the RNA used.

Synthesis of cDNA from human placenta (placental cDNA) anddouble-stranded cDNA (dsDNA)

Before the cDNA synthesis, a check that the placental mRNA containingpoly(A) was intact was carried out in a 1.5% agarose gel.

Then 4 μg of placental mRNA were dissolved in 65.5 μl of H₂O, denaturedat 70° C. for 10 min, and cooled again in ice. The cDNA was synthesizedin a 100 μl mixture after addition of 20 μl of RT₁ buffer (250 mM tris(pH 8.2) at 42° C., 250 mM KCl, 30 mM MgCl₂), 2.5 μl of 20 mM dNTP (i.e.all four deoxynucleoside triphosphates), 1 μl of 1 μg/ml oligo(dT), 1 μlof 1 M DTT, 2 μl of RNAsin and 8 μl of reverse transcriptase (24 U/μl)at 42° C. for 90 min.

Double-stranded cDNA (dsDNA) was synthesized by the method of Gubler andHoffmann (Gene 25 (1983) 263-269). The synthesis was carried outimmediately after the cDNA synthesis by addition of 305.5 μl of H₂O, 80μl of RT₂ buffer (100 mM tris (pH 7.5), 25 mM MgCl₂, 500 mM KCl, 50 mMDTT, 250 μg/ml BSA), 2 μl of RNase H (2 U/μl), 2.5 μl of E. coli DNAligase (5 U/μl), 5 μl of 15 mM β-NAD, and 5 μl of DNA polymerase 1 (5U/μl) and incubation at 15°0 C. for 5 h. The reaction was stopped byheat inactivation (70° C., 30 min).

After addition of 55 μl of 250 μM dNTP, 55 μl of 10 mM tris (pH 7.5), 10mM MgCl₂, 10 μg/ml BSA, 3 μl of T4 DNA polymerase 1 (1 U/μl), 2 μl RNaseH (2 U/μl) and 2 μl of RNase A (2 μg/ml), the reaction mixture wasincubated at 37° C. for a further 30 min in order to correct faultysyntheses of the polymerase I on the second DNA strand (“repairreaction”).

Ligation of EcoRI linkers to the dsDNA, and opening of the linkers

To set up a placental cDNA bank, the dsDNA was provided with EcoRI endsin order to be able to ligate it in the EcoRI cleavage site of the phagevector λgt10 (T. Maniatis et al. (1982), Molecular Cloning, A LaboratoryManual, Cold Spring Harbor). For this purpose of the dsDNA was

a) treated with EcoRI methylase in order to protect internal EcoRIcleavage sites of the dsDNA, and

b) provided with EcoRI linkers which

c) were then opened with EcoRI.

Re a):

The methylase reaction of the dsDNA was carried out immediately afterthe repair reaction by addition of 25 μl of 500 mM EDTA (pH 8.0), 60 μlof methylase buffer (100 mM NaOAc (pH 5.2), 2 mg ofS-adenosyl-L-methionine) and 2 μl of EcoRI methylase (20 U/μl) byincubation at 37° C. for 30 min.

The reaction mixture was extracted with phenol, and the dsDNA wasprecipitated with 60 μl of 4 M NaOAc and 1300 μl of ethanol. The dsDNAwas washed twice with 70% ethanol, extracted once by shaking with ether,and dried.

Re b):

The EcoRI-methylated dsDNA was dissolved in 88 μl of H₂O and, afteraddition of 10 μl of ligase buffer (500 mM tris (pH 7.4), 100 mM MgCl₂,100 mM DTT, 10 mM spermidine, 10 mM ATP, 1 mg/ml BSA) and 1 μl of T4 DNAligase (10 U/μl), was ligated with 1 μl of EcoRI linkers (0.5 μg/μl)(pGGAATTCC and pAGAATTCT) at 15° C. overnight.

Re c):

The volume of the ligase mixture was made up to 120 μl with 6 μl of H₂O,12 μl of 10× EcoRI buffer and 2 μl of EcoRI (120 U/μl). The EcoRIdigestion was carried out at 37° C. for 2 h.

Removal of unbound linkers via a potassium acetate gradient andsize-selection of the dsDNA

To remove all the unbound EcoRI linkers from the dsDNA, the EcoRIreaction mixture was applied in toto to a potassium acetate gradient(5-20% KOAc, 1 mM EDTA, 1 μl/ml ethidium bromide) which was centrifuged(Beckman SW 65/rotor) at 50,000 rpm and 20° C. for 3 h. The gradient wasfractionated from below in such a way that the volume of the first fivefractions was 500 μl, and that of all the remainder was 100 μl. Thefractions were precipitated with 0.01 volume of acrylamide (2 mg/ml) and2.5 volumes of ethanol, washed once with 70% strength ethanol and dried,and each was taken up in 5 μl of H₂O.

To determine the size of the dsDNA, 1 μl of each fraction was analyzedin a 1.5% agarose gel. In addition, the quantity of dsDNA was determinedon 1 μl of each fraction.

Fractions containing dsDNA with over 1000 bp were combined, and thesample was concentrated until the final concentration was 27 μg/ml.

Incorporation of the dsDNA into the phage vector λgt10 and in vitropackaging reaction

The incorporation of the dsDNA into the EcoRI cleavage site of the phagevector λgt10 (Vector Cloning Systems, San Diego, Calif.) was carried outin a 4 μl ligase mixture: 2 μl of dsDNA, 1 μl of λgt10 x EcoRI (1μg/ml), 0.4 μl of ligase buffer, 0.5 μl of H₂O and 0.1 μl of T4 DNAligase. The mixture was incubated at 15° C. for 4 h.

To establish the placental cDNA bank in the phase vector λgt10, theligase mixture was then subjected to an in vitro packaging reaction withthe λ-lysogenic cell extracts E. coli NS 428 and NS 433 at roomtemperature for 2 h (Vector Cloning Systems, San Diego, Calif.; Enquistand Sternberg, Methods in Enzymology 68, (1979), 281-298). The reactionwas stopped with 500 μl of suspensions medium (SM: 0.1 M NaCl, 8 mMMgSO₄, 50 mM tris (pH 7.5), 0.01% gelatine) and 2 drops of chloroform.

Determination of the titer and analysis of the placental cDNA bank

The number of plaque-forming units (PFU) of the placental cDNA bank wasdetermined using competent cells of the E. coli K 12 strain C600 HFL: itwas 1×10⁶ PFU. About 80% of the phages contained DNA inserts larger than1000 base-pairs.

Oligonucleotide probes for screening the placental cDNA bank

Two oligonucleotide probes (20mer probe and 66mer probe) weresynthesized for analysis of the placental cDNA bank. Their sequenceswere deduced from the amino acid primary sequence of several proteolyticand BrCN fragments of F XIIIa. In some cases overlapping, and hencelonger, amino acid sequences were found, and these permitted thesynthesis of a very long probe, namely the 66mer probe.

The 20mer probe is a conventional DNA probe in which all thetheoretically possible codons for the amino acid sequenceMet-Met-Asp-Ile-Thr-Asp-Thr are taken into account (in the case of theterminal amino acid, Thr, the third position in the codon, called the“wobble” position, was omitted; see Table 1). The 20mer probe is thus48-fold degenerate, i.e. a mixture of all the 48 theoretically possiblecoding oligonucleotides for the said amino acid sequence.

The manner of construction and the use of the 66mer probe essentiallyfollowed the rules of Lathe, J. Mol. Biol. 183 (1985) 1-12. In order toconstruct the 66mer probe two 39mer probes were synthesized (39mer Awith the sequence: 5′ TATGGCCAGTTTGAGGATGGCATCCTGGACACCTGTCTG 3′; and39mer B with the sequence: 5′ GTCCATCTGGGCCCGGTCCATCACATACAGACAGGTGTC3′). 39mer A and 39mer B have a complementary sequence comprising 12bases so that hybridization of the two sequences results in long free 5′ends.

The two 39mer probes were (as was the 20mer probe) labeled at the 5′ endwith T4 polynucleotide kinase in the presence of (γ-³²P)-ATP (about 1 μgof DNA, (γ-³²P)-ATP: 3000 Ci/mmol, 10 μCi/μl, with 6 μl/40 μl reactionmixture being used). The 20mer probe had a specific activity of 1×10⁸Bq/μg or 1.5×10⁶ Bq/pmol. The two 39mer probes were heated at 95° C. for5 min., mixed and slowly cooled to 4° C. in a cold room, and thushybridized. Then about 1 μg of the hybridized 39mer probes was treatedwith DNA polymerase I, Klenow fragment, with the addition of(α-³²P)-dATP (3000 Ci/mmol, 10 μCi/μl, 4 μl/50 μl reaction mixture)(5′→3′ filling-in reaction). The filled-in 66mer probe had a specificactivity of 1.5×10⁸ Bq/μg. The DNA probes were stored at −20° C.; the20mer probe was used immediately for analysis (screening), while the66mer probe had previously been heated at 95° C. for 5 min and thenrapidly cooled in an ice bath.

Since the 66mer probe had been produced by hybridization of two 39mersfollowed by a 5′→3′ filling-in reaction, it is possible to carry outvarious experiments. On the one hand, it is possible to hybridize cDNAbanks with the denatured 66mer DNA probe and, on the other hand, it isthen possible to hybridize positive clones with the 39mer A and B probesindividually.

It is highly probable that the clones which hybridize both with the longprobe and with both short 39mer probes A and B have the desiredsequence. Thus, the method of constructing a long, complexoligonucleotide probe and of “rescreening” the clones using thepartially complementary short DNA probes, which has been described,represents an enhancement of specificity. In addition, it is possiblewith the long oligonucleotide and its partially complementary shortpartoligonucleotides to screen genomic bands with enhanced specificity.Another advantage of the said method is that the synthesis of shorteroligonucleotides can be carried out more easily and with higher yieldsand accuracy. The sequence of two enzymatic reactions, namely 1) T4polynucleotide kinase for the (γ-³²P)-ATP labeling, and 2) DNApolymerase filling-in reaction with addition of (α-³²P)-dNTP, means thatit is possible to obtain higher specific activities (at least 1×10⁸Bq/μg DNA).

Screening of the placental cDNA with F XIIIa-specific oligonucleotides

5×10⁵ PFU of the placental cDNA bank were examined for cDNA sequencescoding for F XIIIa using the 20mer probe and the 66mer probe. Thisentailed 3×10⁴ PFU being plated out with cells of the E. coli K 12strain C 600 HFL in soft agar in 13.5 cm Petri dishes and incubated at37° C. for 6 h. Any lysis which had taken place by this time was stillincomplete. The plates were incubated in a refrigerator overnight, andthe phages were transferred to nitrocellulose filters (Schleicher &Schüll, BA 85, Ref. No. 401124) (duplicates). The nitrocellulose filtersand Petri dishes were marked with an injection needle in order to allowsubsequent allocation. The Petri dishes were stored in a cold roomduring the processing of the nitrocellulose filters. The DNA on thenitrocellulose filters was denatured by placing the filters for 5 min.on filter paper (Whatman M 3) impregnated with 1.5 M NaCl, 0.5 M NaOH.The filters were then renatured in the same way using 1.5 M NaCl, 0.5 Mtris (pH 8.0) and washed with 2×SSPE (0.36 M NaCl, 16 mM NaOH, 20 mMNaH₂PO₄, 2 mM EDTA). The filters were then dried in vacuo at 80° C. for2 h. The filters were washed in 3×SSC, 0.1% SDS (20×SSC=3 M NaCl, 0.3 MNa citrate) at 65° C. for 4 h, and prehybridized at 65° C. for 4 h(prehybridization solution: 0.6 M NaCl, 0.06 M tris (pH 8.3), 6 mM EDTA,0.2% non-ionic synthetic sucrose polymer ({circle around (R)}Ficoll),0.2% polyvinylpyrrolidone 40, 0.2% BSA, 0.1% SDS, 50 μg/ml denaturedherring sperm DNA). The filters were incubated overnight with theaddition of 100,000-200,000 Bq of the labeled oligonucleotide per ml ofhybridization solution (as prehybridization solution but without herringsperm DNA) in beakers or in sealed polyethylene films, shaking gently.The hybridization temperature for the 20mer probe and for the 39merprobes was 42° C., and that for the 66mer probe was 47° C.

The nitrocellulose filters were washed with 6×SSC, 0.05 M sodiumpyrophosphate at room temperature for 1 h and at the particularhybridization temperature for a further hour. The filters were dried andautoradiographed overnight. Signals occurring on both duplicate X-rayfilms were allocated to the Petri dish, and the region (about 50plaques) was punched out with the wide end of a Pasteur pipette, and thephages were re-suspended in 1 ml of SM buffer. Positive phages weresingled out over three rounds until a single clone was obtained.

A total of 5×10⁵ PFU of the placental cDNA bank was examined in severalpassages. 17 signals were identified on duplicate filters. Furtherscreening under more stringent conditions resulted in 7 signals stillbeing positive. Of these 7 PFU only one PFU showed a positive signalboth after hybridization with the 20mer and 66mer probes and with the39mer probes A and B. This clone—called λgt10-12 hereinafter—has asequence of 1704 base-pairs coding for F XIIIa and having an internalEcoRI cleavage site. Southern blot analysis shows that the smaller EcoRIfragment of 540 base-pairs hybridizes with the 20mer DNA probe, and thelarger fragment of 1164 base-pairs hybridizes with the 66mer DNA probe.

On rescreening, it emerged from the Southern blot that there is morereaction with the 39mer probe A than with the 39mer probe B. Sequenceanalysis of the clone λgt10-12 showed subsequently that, over the entirelength of the 66mer probe, there are only seven mismatches to thesequence found for F XIIIa (Table 2). The seven mismatches aredistributed as follows: there are three in the 39mer A probe and fivemismatches in the 39mer B probe (one mismatch occurs in the overlappingregion, and thus is common to both). The five mismatches in the 39mer Bprobe are clustered, which is possibly the reason for the weakerhybridization signals in the case of the 39mer B probe.

Screening of the placental cDNA with nick-translated EcoRI fragments

The two subcloned EcoRI fragments, which were 540 base-pairs and 1164base-pairs in length, were cloned into the EcoRI cleavage site of thecommercially available vector pUC8 (1164 bp=pUC8-12.1 and 540bp=pUC8-12.2) and were isolated therefrom preparatively with EcoRI, andnick-translated in the presence of (α-³²P)-dNTP. The specific activityof both fragments was 1×10⁸ Bq/μg DNA. Using the (³²P)-labeled fragmentsin several passages, about 1×10⁶ recombinant phages of the placentalcDNA bank were examined (hybridization temperature 65° C.) and thus 13hybridizing phages were identified. The phages were singled out overthree rounds until a single homogeneous phage preparation was obtained.20 ml lysates of each phage were set up, and the DNA was extracted. TheDNA was digested with EcoRI and fractionated on a 1% agarose gel. Thegel was subjected to the Southern blot technique, and the nitrocellulosefilter was hybridized with the labeled 540 bp EcoRI fragment. Elevenphages showed hybridization signals. The nitrocellulose filter wasboiled and hybridized with the nick-translated 1164 bp EcoRI fragment.Nine phages showed hybridization signals.

It was possible to identify, on the basis of the size of the hybridizingfragments, clones which, in comparison with λgt10-12, expand bothtowards the 5′ end such as λgt10-20 and towards the 3′ end such asλgt10-11. It was possible by use of these clones to determine thecomplete F XIIIa cDNA sequence. It was possible to combinepart-sequences which were present by use of internal restriction sitesor by hybridization of overlapping sequences. The complete cDNA sequencecan be ligated into expression vectors and expressed in suitableprokaryotic or eukaryotic systems.

DNA sequence analysis

The phage clone λgt10-12 was multiplied, and the DNA was extracted. Thetwo EcoRI fragments were isolated and cloned into the EcoRI site of theplasmid vector pUC8. pUC8-12.1 has the 1164 base-pair fragment, andpUC8-12.2 has the 540 base-pair fragment. In order to isolate the entirefragment comprising 1704 base-pairs, λgt10-12 was partially digestedwith EcoRI, and the 1704 base-pair band was isolated and cloned into theEcoRI site of pIC19H (Marsh et al., Gene 32 (1984) 481-485). Theresulting plasmid is called pIC19H-12.

It was possible by cloning Sau 3A, AluI and TaqI sub-fragments of theclones pUC8-12.1, pUC8-12.2 and pIC19H-12 into pUC plasmids and M13phages, followed by sequencing of the relevant regions using theenzymatic dideoxy method of Sanger and the chemical method of Maxam andGilbert, to determine the sequence of the 1704 bp fragment (Table 3).The sequence shows only one open reading frame and codes for the first542 amino acids of the factor XIIIa molecule.

Restriction analysis of the clone λgt10-12 or pIC19H-12 and of theclones λgt10-11 and λgt10-20 was carried out both by suitable single andmultiple digestions and by partial digestion of (³²-P)-labeled DNAfragments by the method of Smith and Birnstiel (Smith, H. O. andBirnstiel, M. L., Nucleic Acids Res. 3 (1976) 2387-2398) (FIG. 1).

The clone λgt10-11 has a fragment which is 2432 bp in size and has aninternal EcoRI cleavage site. This fragment overlaps by 237 bp at the 3′end the cDNA fragment from λgt10-12, and comprises the remaining 570 bpof the coding sequence plus 1625 bp of the non-coding region including apoly(A) sequence of 89 bases. The clone λgt10-20 with a cDNA fragmentabout 700 bp in size also has at the 5′ end 6 bases more (GAG GAA . . .) than λgt10-12.

2. Preparation of a clone which can be expressed and contains the entirecDNA coding for F XIIIa

The starting clones λgt10-11 and λgt10-12 were used to obtain a plasmidwhich contains the entire coding region of the F XIIIa cDNA. With theaid of partial EcoRI digestion, the insertion, comprising 1704base-pairs, of λgt10-12 was cloned into the EcoRI site of pIC19H (Marshet al., loc. cit.). The resulting plasmid pIC19H-12.1 was usedsubsequently (see FIG. 2). The clone λgt10-11 has an insertion 2432base-pairs in size and has an internal EcoRI cleavage site. The left(“5′-terminal”) EcoRI fragment, which is 1224 base-pairs in size andembraces the C-terminal 570 base-pairs of the coding sequence plus 654base-pairs of the 3′ non-coding region, was likewise cloned into theEcoRI cleavage site of pIC19H. The resulting plasmid pIC19H-11.1 wasused subsequently (FIG. 2). The plasmids pIC19H-12.1 and pIC19H-11.1have the coding region for F XIIIa in the same orientation in the vectorand have an overlapping region which comprises 237 base-pairs. Thisoverlapping region was used to construct from the part-clonespIC19N-12.1 and pIC19H-11.1 a clone which embraces the entire codingregion. This entailed preparation, from the two plasmids mentioned, ofpartially single-stranded heteroduplex molecules by hybridization invitro (FIG. 2) and transformation of the reaction mixture into E. coli.By utilization of the repair mechanisms of the bacterium (3′→5′exonuclease activity, 5′→3′ polymerization activity of the enzyme DNA plymerase I), a plasmid with the entire coding region was obtained.

Specifically, this entailed 1 μg of the DNA of each of the plasmidspIC19H-12.1 (ClaI-digested) and pIC19H-11.1 (BamHI-digested) being mixedand precipitated with ethanol. The DNA was dried in vacuo, taken up in20 μl of H₂O and, after addition of 5 μl of 1 N NaOH, incubated at roomtemperature for 10 minutes. The following were then added in thesequence indicated: 200 μl of H₂O, 25 μl of 1 M tris.HCl (pH 8.0) and 50μl of 0.1 N HCl. The reaction mixture was incubated at 65° C. for 3hours, precipitated with ethanol, and dried. The DNA was resuspended in20 μl of H₂O and transformed by known methods into E. coli, and thecells were plated onto LB-amp plates and incubated overnight. Of thetotal of 96 ampicillin-resistant clones, 24 clones were worked up by thealkali method (Birnboim and Doly, Nucl. Acid Res. 7 (1979) 1513-1523),and the plasmids were characterized by restriction endonucleolysis withHindIII, EcoRI, BamHI and PvuII. Six plasmids showed the expectedrestriction pattern. One of these plasmids, pFXIII-13 (FIG. 2), wascharacterized in detail. This entailed the StuI (position 1225)-PvuII(position 1870) fragment, which embraces the 237 base-pair overlappingregion, being sequenced, and the DNA sequence was confirmed as correct.The plasmid pFXIII-13 comprises 2693 base-pairs of FXIIIa cDNA, of which78 bp are of the 5′ non-translated region, 2196 bp are the entire codingregion, and 419 bp are of the 3′ non-translated region. pFXIII-13 wasthe starting plasmid for all subsequent expression experiments.

3. Expression of biologically active factor XIIIa in E. coli

a) Construction of F XIIIa expression plasmids

pFXIII-13 has the F XIIIa cDNA insert in the correct orientation withrespect to the lac promoter and in the correct reading frame withrespect to the lacZ α-peptide. pFXIII-13 is thus able to induce thesynthesis of a F XIIIa fusion protein in E. coli. The molecular weightof this protein comprises the 732 amino acids of natural F XIIIatogether with 16 vector-coded amino acids plus 28 amino acids specifiedby the 5′ non-coding region. These additional 44 amino acids are locatedat the N-terminal end of the fusion protein. The expected molecularweight of this protein is 85,250 D (Tab. 4).

An expression plasmid which uses in place of the lac promoter a moreefficient trp/lac hybrid promoter was subsequently constructed. Forthis, pKK233-2 (Amann and Brosius, Gene 40 (1985), 183-190) was cut withEcoRI and treated with Bal31 (FIG. 3). The DNA was then cut with PvuII,and the fragment 2800 base-pairs in size was purified on a PAA gel. Thisfragment was religated in the presence of a BglII linker (5′-CAGATCTG).The resulting plasmid pTrc89-1 (FIG. 3) was cut with NcoI and HindIII,and the synthetic linker

5′ CATGGAATTCGA 3′

3′ CTTAAGCTTCGA 5′

was incubated together with the 2800 base-pair fragment in a ligasemixture. The resulting plasmid, pTrc96A (FIG. 3), was cut with EcoRI andHindIII, and the fragment 2800 base-pairs in size was gel-purified andligated with the EcoRI-HindIII linker which is 55 base-pairs in sizefrom pUC18. The resulting plasmid is pTrc97A (FIG. 3). In contrast topKK233-2, pTrc97A has, downstream of the NcoI site which occurs onlyonce in the plasmid, the polylinker from pUC18 and thus has numerouscloning sites.

The FXIIIa cDNA cloned into pFXIII-13 has a PstI site 21 base-pairs 5′away from the ATG initiation codon (position 61). The next PstI site islocated in the 3′ untranslated region (position 2398). The PstI fragmentwhich is 2337 base-pairs long was isolated from pFXIII-13 and ligatedinto the PstI site of pTrc97A. The resulting plasmid with the PstIfragment in the desired orientation is pFXIII-C4 (FIG. 3 a). Theexpected FXIII molecule specified by pFXIII-C4 has 22 additionalN-terminal amino acids, 15 of them being vector-coded and 7 beingspecified by the 5′ non-coding region of the F XIII cDNA. The expectedmolecular weight of this protein is 82,830 D (Tab. 4).

Use of thrombin to cleave off the 37 amino-terminal amino acids convertsthe F XIIIa into the active form. Since such a F XIIIa molecule whichhas already been activated is of therapeutic interest, an attempt wasmade to express in E. coli a F XIIIa that can be shortened by thrombincleavage. In order to obtain a high yield the cloning was carried out insuch a way that the shortened F XIIIa is expressed in the form of ahybrid protein, fused to an E. coli β-galactosidase fragment. TheSmaI-HindIII fragment which is about 2700 bp in size from pFXIII-13 wasisolated and ligated into pBD21C20H which had been hydrolyzed with SmaIand HindIII. In the new plasmid pMB259 (FIG. 3 b), the coding region ofthe cDNA for F XIIIa from amino acid Pro₃₇ to Met₇₃₂ is located in thereading frame applying to the 375 aminoterminal amino acids of theβ-galactosidase, and it has the thrombin cleavage site Arg₃₈/Gly₃₉ sothat it is possible to obtain activated F XIIIa by thrombin cleavagefrom the synthesized fusion proteins.

The expression vector pBD2IC20H had been constructed by ligating thepolylinker region, which comprises 58 bp, of the plasmid pIC20H (Marshet al., loc. cit.) as BamHI-HindIII fragment in pBD2 (Broker, Gene Anal.Techn. 3 (1986) 53-57) which has the lac promoter.

b) Expression

It was found that E. coli cells of the strain D29A1 which aretransformed with pFXIII-13, with pFXIII-C4 or with pMB259 are able tosynthesize the expected F XIIIa proteins. The expression of F XIII bythe plasmids pFXIII-13, pMB 259 and pFXIII-C4 can be induced with IPTG.Comparison of protein extracts after IPTG induction of E. coli D29A1(pFXIII-13) and D29A1 (pFXIII-C4) on PAA gels stained with Coomassieblue showed the expected molecular weights of the F XIII fusionproteins. The estimated expression of the “44aa FXIII fusion protein” isabout 5 times that of the “22aa FXIII fusion protein”.

It was also found that the F XIIIa molecules specified by pFXIII-13 andpFXIII-C4 have biological activity. In contrast, no F XIII activity wasfound in E. coli D29A1 control extracts. The activity found in the clotstability assay (Karges, in Bergmeier, Methods of Enzymatic Analysis,Volume 5, Enzymes 3: Peptidases, Proteinases and their Inhibitors, pages400-410) is 5 μg/l for E. coli D29A1 (pFXIII-13), based on the E. coliculture (0D₂₅₀=1.5), and is 15 μg/l for D29A1 (pFXIII-C4).

The amount of factor XIII found in the F XIIIa-specific ELISA is 1.5mg/l, based on the E. coli culture, for pFXIII-13 and is 3 mg/l forpFXIII-C4. The discrepancy between the amounts of F XIII measured in thebiological assay and in the ELISA derives from the fact that the majorpart (>90%) of F XIII in the E. coli cell is in the form of an insolubleprecipitate which is biologically inactive and dissolves only in 7 Murea. The soluble fraction of the F XIII molecules present in E. coliextracts shows in the Ouchterlony test (Ouchterlony, Progr. Allergy 5(1958) 1) a precipitation curve which is substantially identical to thatof F XIII isolated from placenta.

The expression of eukaryotic proteins in E. coli in the form ofinsoluble protein aggregates has already been described for severalproteins. These proteins can be dissolved out of such aggregates using achaotropic agent and can be converted by suitable renaturing conditionsinto their biologically active form.

4. Expression of F XIII in yeasts

The synthesis of biologically active F XIII obtained by genemanipulation from yeasts was achieved by incorporating the cDNA codingfor F XIII into expression vectors which are able to replicateautonomously in yeasts. It was possible to isolate F XIII-active proteinfrom extracts of the recombinant clones.

The conditions for growing yeasts and the molecular biological methodsare described in Dillon et al., Recombinant DNA Methodology, John Wiley& Sons, New York (1985) and in Maniatis et al., loc. cit.

The F XIII cDNA was isolated from the vector pFXIII-13 as a HindIIIfragment about 2700 bp in size, and was cloned into the HindIII site ofthe vector pAAH5 (Ammerer, Meth. Enzymol. 101 (1983), 192-201). Thus,the F XIII cDNA in the resulting plasmid pMB240 (FIG. 4) is under thecontrol of the strong ADHI promotor which contains the gene expressionsignals of alcohol dehydrogenase. The plasmid pMB240 was transformedinto baker's yeast, Saccharomyces cerevisiae, strain Leu 2-3, Pep 4-3,by the method of Itoh et al. (J. Bacteriol. 153 (1983), 163-168) andLeu⁺ transformants were selected on YNB minimal medium. One colony oftransformed yeast cells was used to inoculate a liquid culture with YNBmedium. After growth at 30° C. for two days, transfer into complex YPBmedium was carried out and, after a further three days, the cells wereremoved by centrifugation and were disrupted with a glass bead mill inisotonic saline solution containing 100 mM sodium citrate (pH 7.2). Thecell extract was subjected to high-speed centrifugation in a Sorvallhigh-speed centrifuge, in a SS34 rotor, at 20,000 rpm and 4° C. for 1hour.

The cell-free supernatant of S. cerevisiae (pMB240) was analyzed by theWestern blot method. In addition to a band which can be detected in thesame position as F XIII from placenta, a protein of about 116,000 Dreacted specifically with the anti-F XIII serum used. This proves thatpart of the F XIII formed in yeasts is glycosylated. The glycosylationof proteins is often a factor prolonging the half-life of proteins,especially plasma proteins. In addition, carbohydrate side-chains mayincrease the activity or extend the duration of the action of plasmaproteins, for example antithrombin III. A F XIII which is expressed inyeast and which, in contrast to F XIII obtained from placenta, isglycosylated or has undergone posttranslatimal modification in someother way can have, owing to an increased activity, advantages over FXIII from placenta.

The cell-free supernatant was examined for F XIII by an ELISA, and the FXIII concentration was found to be 150 ng/ml, based on the yeastculture. The biological activity of F XIII was determined by the methodof Karges, loc. cit., and confirmed the concentrations measured in theELISA. It was possible to rule out a non-specific F XIII-like activityby yeast proteins because the biological activity of the F XIII obtainedfrom baker's yeast could be specifically inhibited by anti-F XIIIantibodies.

5. Expression of F XIIIa in animal cells

a) Construction of expression vectors for animal cells

The expression vector pSVA STOP1 is proposed in German PatentApplication P 36 24 453.8 (Example 1 of this application, which relatesto the synthesis of this vector, has been extracted and is reproduced inthe Appendix). Apart from this plasmid, use was made of the vectorspZET4 (see below) and pSP6HS9, which has the Drosophila heat shockprotein 70 promotor (Wurm et al., Proc. Natl. Acad. Sci. USA 83 (1986)5414-5418), for the expression of F XIIIa in animal cells.

pZET4 (FIG. 5): the plasmid pSVA STOP1 was cut with BamHI, and thevector fragment which is 2.6 kb in size and has the SV40 early promotorwas isolated. The BglII-BamHI fragment 0.85 kb in size from pSV2dhfr(Lee et al., Nature 294 (1981) 228-232) was ligated into the vectorwhich had been pretreated in this way, which resulted in the plasmidpZET4. Located on the 0.85 kb fragment from pSV2dhfr are mRNA splicesites from exon-intron joins, and the polyadenylation site of the genefor the t antigen from the SV40 DNA.

b) Construction of F XIIIa expression vectors for animal cells

pSVF13 (FIG. 5 a): the expression vector pSVA STOP1 was cut with HindIIIand XbaI. A HindIII-XbaI fragment, about 2.7 kb in size and having the FXIIIa cDNA, from pFXIII-13, was ligated into the vector which had beentreated in this way. The F XIIIa transcription unit on pSVF13 has nomRNA splice sites.

pZF13 (FIG. 5 b): a HindIII fragment about 2.7 kb in size and containingthe F XIIIa cDNA was isolated from the plasmid pFXIII-13. The resulting5′ protruding end was eliminated by filling in the complementary strandwith DNA polymerase I (Klenow fragment). The expression vector pZET4 waslinearized by cutting at the unique XbaI site. The resulting 5′protruding end was likewise eliminated by filling in the complementarystrand with DNA polymerase I (Klenow fragment). Ligation of thefilled-in vector with the filled-in F XIIIa cDNA fragment results in theF XIIIa expression plasmid pZF13. As in pSVF13, the F XIIIa cDNA isunder the transcriptional control of the SV40 early promoter but isprovided with mRNA splice sites.

pHSF13 (FIG. 5 c): the plasmid pSVF13 was partially digested with EcoRI,and a fragment about 2.9 kb in size and having the F XIIIa cDNA followedby the SV40 polyadenylation site for early transcripts was isolated.This fragment was ligated into the unique EcoRI site of the plasmidpSP6HS9 downstream of the heat shock protein 70 promotor.

c) DHFR expression vectors for the cotransfection of CHO (Chinesehamster ovary) dhfr⁻ cells

Either the DHFR vector pSV2dhfr (Lee et al., loc. cit.) or thepromotorless DHFR plasmid pSV0Adhfr (German Patent Application P 36 24453.8, see Appendix) was used for the cotransfection with the describedF XIIIa expression vectors in CHO dhfr⁻ cells. Both vectors have theDHFR cDNA from the mouse.

d) Vector conferring G418 resistance for the cotransfection of BHK (babyhamster kidney) cells

The F XIIIa expression vectors which have been described werecotransfected with the vector pRMH140 (Hudziak et al., Cell 31 (1982),137-146) in BHK cells.

e) Expression of F XIIIa in CHO cells

Cotransfection of the plasmid pZF13 with the DHFR vector pSV2dhfr, andof the expression plasmid pSVF13 together with the DHFR vectorpSVOAdhfr, was carried out using the calcium phosphate precipitationmethod (Graham and van der Eb, Virology 52 (1973), 456-467) in CHO dhfr⁻cells. This entailed 20 μg of the particular F XIIIa expression plasmid(pZF13 or pSVF13) being mixed and coprecipitated with 5 μg of the DHFRvectors (pSV2dhfr or pSVOAdhfr). The coprecipitate was used fortransfection as described above (0.5×10⁶ cells in a 25 cm² culturebottle). After 3 days, the cells were trypsinized, transferred intothree 60 mm Petri dishes and mixed with selection medium (containing noglycine, hypoxanthine or thymidine). The only cells which survive underthese conditions are those which have undergone stable transfection withthe DHFR gene. Colonies of transfected cells become visible on the Petridishes after 1-3 weeks. The following transfection rates were achievedby this method:

-   -   pSV2dhfr 5×10⁻⁵/plate    -   pSVOAdhfr 1×10⁻⁵/plate        Individual clones were isolated and multiplied in a medium        containing no glycine, hypoxanthine or thymidine.

A specific ELISA with a lower detection limit of about 3 ng/ml was usedto detect F XIIIa in culture supernatants and cell lysates of theindividual clones. Culture supernatants were used as such in the ELISA.The cell lysates were prepared as follows: Confluent cells in 25 cm²culture bottles were washed twice in 40 mM tris.HCl (pH 7.4), 1 mM EDTA,150 mM NaCl, taken up in 150 μl of 0.25 M tris.HCl (pH 7.8), 5 mM DTT,2% glycerol, 0.2% detergent (Triton X100), and lyzed by freezing andthawing three times.

In soluble constituents of the cells were removed by centrifugation. Thelysate was diluted 1:2.5 for use in the ELISA. The detection methodwhich has been described was applied to 17 clones for the combination ofthe plasmids pZF13/pSV2dhfr, and one clone expressing F XIIIa (CH059-5-C7) was found. After cotransfection with the plasmidspSVF13/pSVOAdhfr and analysis of 12 clones, a further F XIIIa-expressingclone (CH0 60-3-C1) was detected. With both the positive clones it waspossible to detect F XIIIa in the medium and in the lysate in the samerelative amounts. In order to determine quantitatively the expressionrate of the lines producing F XIIIa the following standard procedure wascarried out:

0.5×10⁶ cells were plated out in 5 ml medium in 25 cm² culture bottles.The medium was changed after 24 hours (5 ml). Another 24 hours later themedium was removed, the cell count was determined, and lysates wereprepared. For all the expression rates (ng/10⁶ cells/24 h) statedhereinafter, the cell count per 25 cm² bottle at the end of the test was1±0.25×10⁶ cells. The table which follows shows the expression rate ofthe basic clones tested in the manner described:

extracellular intracellular (ng/10⁶ cells/ (ng/10⁶ cells/ 24 h) 24 h)CHO 59-5-C7 12  9 CHO 60-3-C1 17 13

On SDS electrophoresis followed by F XIIIa-specific immunoblotting oflysates and supernatants of the clone CH0 59-5-C7 and the clone CH060-3-C1, in each case one band with the molecular weight of the proteinisolated from human placenta showed a reaction.

The clone CH0 59-5-C7 was exposed to increasing concentrations ofmethotrexate (Mtx) for gene amplification. Starting with a concentrationof 10 nM Mtx and a 4-transfer adaptation time, the Mtx concentration inthe medium was increased to 50 nM. The following expression rates weredetermined in the standard procedure:

extracellular intracellular Mtx (nM) (ng/10⁶ cells/24 h) (ng/10⁶cells/24 h) 0 12 9 10 19 16 50 38 66f) Expression of F XIIIa in BHK cells

20 μg of each of the F XIIIa expression plasmids pZF13, pSVF13 andpHSF13 were cotransfected with 5 μg of the plasmid pRMH140 which codesfor G418 resistance, by the calcium phosphate precipitation methoddescribed in example 5e) in BHK cells. After 3 days, the cells weretrypsinized, transferred into three 60 mm Petri dishes and mixed withselection medium containing 400 μg/ml G418. After 12 days about 200-300G418 resistant colonies had grown in each Petri dish. The total numberof clones was trypsinized and subjected to transfers as combined clone(CC) in 25 cm² culture bottles (5 ml of medium).

In the case of cells transfected with pZF13 and pSVF13, where an 80-100%confluence had been reached F XIIIa was determined in the medium and inthe relevant lysate (see example 5e)) using a specific ELISA. Combinedclones which had been transfected with pHSF13 and had likewise reached80-100% confluence were mixed with fresh medium which had been preheatedto 42° C. and were incubated at 42° C. for one hour. After anotherreplacement of the medium with fresh medium equilibrated at 37° C., thecells were maintained at 37° C. for 24 hours. The medium and lysateswere then examined for their content of F XIIIa as described above. Thetable which follows summarizes the cellular distribution of F XIIIa forthe various combined clones.

extracellular intracellular (ng) (ng) BHK-MK1 (pZF13) 65 22 BHK-MK2(pZF13) 80 20 BHK-MK3 (pSVF13) 85 18 BHK-MK5 (pHSF13) 170 11

The expression rates relating to the F XIIIa present in the medium weredetermined for the individual combined clones by the standard proceduredescribed in example 5e). For all the expression rates (ng/10⁶ cells/24h) stated hereinafter, the cell count per 25 cm² bottle at the end ofthe test was 4.5±0.5×10⁶ cells.

extracellular (ng/10⁶ cells/24 h) BHK-MK1 (pZF13) 3.4 BHK-MK2 (pZF13)5.6 BHK-MK5 (pHSF13) 3.8 BHK-MK6 (pHSF13) 3.8

Since the transfected BHK lines described hitherto have been mixedpopulations including cells which were not producing or differed intheir expression rates, it was subsequently attempted to isolategenetically uniform cell lines with high expression rates by singlingout clones. For this purpose, cells from the particular combined clonewere placed on microtiter plates in a concentration of 1 cell/well, 2cells/well or 4 cells/well. Supernatants from wells in which only oneclone had grown were analyzed by the F XIIIa-specific ELISA. The cloneswith the highest expression rates were multiplied in 25 cm² culturebottles, and their expression rates were investigated by the standardprocedure described above. The table which follows shows the expressionrate of clones obtained by singling out BHK-MK1 (pZF13) in the mannerdescribed.

extracellular (ng/10⁶ cells/24 h) BHK MK1 (pZF13) 3.4 BHK MK1-A12(pZF13) 8 BHK MK1-E2 (pZF13) 25 BHK MK1-F12 (pZF13) 14 BHK MK1-C1(pZF13) 22

It was also shown, taking the example of the BHK cell line MK1-E2, thatthe F XIIIa molecules synthesized by these cells have biologicalactivity. 10⁸ cells of the BHK MK1-E2 line and of the non-transfectedBHK line which was used (negative control) were taken up in 1.5 ml of0.25 M tris.HCl (pH 7.8) containing 2% glycerol. The cells were lyzed byfreezing and thawing three times, followed by treatment in a Douncehomogenizer. After removal of insoluble constituents by centrifugation,the lysate was used in the biological assay (see example 3). Thefollowing F XIIIa activities were found:

BHK MK1-E2 0.06 units/10⁸ cells BHK (not transfected) 0 units/10⁸ cells

TABLE 1 20mer probe, 48-fold degenerate Amino acid sequence Met Met AspIle Thr Asp Thr DNA probe ATG ATG GAT ATT ACT GAT AC C C A C A C G

TABLE 2 66mer probe, non-degenerate Amino acid # 1 2 3 4 5 6 7 8 9 10 11Amin acid Tyr Gly Gln Phe Glu Asp Gly Ile Leu Asp Thr sequence PossiblemRNA UAU GGU CAA UUU GAA GAU GGU AUU UUA GAU ACU sequences UAC GGC CAGUUC GAG GAC GGC AUC UUG GAC ACC GGA GGA AUA CUU ACA GGG GGG CUC ACG CUACUG 66mer probe TAT GGC CAG TTT GAG GAT GGC ATC CTG GAC ACC Sequencfound TAT GGT CAG TTT GAA GAT GGC ATC CTG GAC ACT Amino acid # 12 13 1415 16 17 18 19 20 21 22 Amin acid Cys Leu Tyr Val Met Asp Arg Ala GlnMet Asp sequence Possible mRNA UGU UUA UAU GUU AUG GAU CGU GCU CAA AUGGAU sequences UGC UUG UAC GUC GAC CCG GCG CAG GAC CUU GUA CGA GCA CUCGUG CGG GCG CUA AGA CUG AGG 66mer probe TGC CTG TAT GTG ATG GAC CGG GCCCAG ATG GAC Sequenc found TGC CTG TAT GTG ATG GAC AGA GCA CAA ATG GAC

TABLE 3     1GAGGAAGTCCCCGAGGCGCACAGAGCAAGCCCACGCGAGGGCACCTCTGGAGGGGAGCGCCTGCAGGACCTTGTAAAGTC   81 AAAA METSER GLU THR SER ARG THR ALA PHE GLY GLY ARG ARG ALA VAL PRO PRO ASN ASN     ATG TCA GAA ACT TCC AGG ACC GCC TTT GGA GGC AGA AGA GCA GTT CCA CCCAAT AAC   142 SER ASN ALA ALA GLU ASP ASP LEU PRO THR VAL GLU LEU GLN GLY VAL VAL PRO ARGGLY  TCT AAT GCA GCG GAA GAT GAC CTG CCC ACA GTG GAG CTT CAG GGC GTG GTGCCC CGG GGC   202  VAL ASN LEU GLN GLU PHE LEU ASN VAL THR SER VAL HISLEU PHE LYS GLU ARG TRP ASP  GTC AAC CTG CAA GAG TTT CTT AAT GTC ACG AGCGTT CAC CTG TTC AAG GAG AGA TGG GAC   262  THR ASN LYS VAL ASP HIS HISTHR ASP LYS TYR GLU ASN ASN LYS LEU ILE VAL ARG ARG  ACT AAC AAG GTG GACCAC CAC ACT GAC AAG TAT GAA AAC AAC AAG CTG ATT GTC CGC AGA   322  GLYGLN SER PHE TYR VAL GLN ILE ASP LEU SER ARG PRO TYR ASP PRO ARG ARG ASPLEU  GGG CAG TCT TTC TAT GTG CAG ATT GAC CTC AGT CGT CCA TAT GAC CCC AGAAGG GAT CTC   382  PHE ARG VAL GLU TYR VAL ILE GLY ARG TYR PRO GLN GLUASN LYS GLY THR TYR ILE PRO  TTC AGG GTG GAA TAC GTC ATT GGT CGC TAC CCACAG GAG AAC AAG GGA ACC TAC ATC CCA   442  VAL PRO ILE VAL SER GLU LEUGLN SER GLY LYS TRP GLY ALA LYS ILE VAL MET ARG GLU  GTG CCT ATA GTC TCAGAG TTA CAA AGT GGA AAG TGG GGG GCC AAG ATT GTC ATG AGA GAG   502  ASPARG SER VAL ARG LEU SER ILE GLN SER SER PRO LYS CYS ILE VAL GLY LYS PHEARG  GAC AGG TCT GTG CGG CTG TCC ATC CAG TCT TCC CCC AAA TGT ATT GTG GGGAAA TTC CGC   562  MET TYR VAL ALA VAL TRP THR PRO TYR GLY VAL LEU ARGTHR SER ARG ASN PRO GLU THR  ATG TAT GTT GCT GTC TGG ACT CCC TAT GGC GTACTT CGA ACC AGT CGA AAC CCA GAA ACA   622  ASP THR TYR ILE LEU PHE ASNPRO TRP CYS GLU ASP ASP ALA VAL TYR LEU ASP ASN GLU  GAC ACG TAC ATT CTCTTC AAT CCT TGG TGT GAA GAT GAT GCT GTG TAT CTG GAC AAT GAG   682  LYSGLU ARG GLU GLU TYR VAL LEU ASN ASP ILE GLY VAL ILE PHE TYR GLY GLU VALASN  AAA GAA AGA GAA GAG TAT GTC CTG AAT GAC ATC GGG GTA ATT TTT TAT GGAGAG GTC AAT   742  ASP ILE LYS THR ARG SER TRP SER TYR GLY GLN PHE GLUASP GLY ILE LEU ASP THR CYS  GAC ATC AAG ACC AGA AGC TGG AGC|TAT GGTCAG TTT GAA GAT GGC ATC CTG GAC ACT TGC   802  LEU TYR VAL MET ASP ARGALA GLN MET ASP LEU SER GLY ARG GLY ASN PRO ILE LYS VAL  CTG TAT GTG ATGGAC AGA GCA CAA ATG GAC CTC TCT GGA AGA GGG AAT CCC ATC AAA GTC                                  66 mer|   862  SER ARG VAL GLY SER ALAMET VAL ASN ALA LYS ASP ASP GLU GLY VAL LEU VAL GLY SER  AGC CGT GTG GGGTCT GCA ATG GTG AAT GCC AAA GAT GAC GAA GGT GTC CTC GTT GGA TCC   922 TRP ASP ASN ILE TYR ALA TYR GLY VAL PRO PRO SER ALA TRP THR GLY SER VALASP ILE  TGG GAC AAT ATC TAT GCC TAT GGC GTC CCC CCA TCG GCC TGG ACT GGAAGC GTT GAC ATT   982  LEU LEU GLU TYR ARG SER SER GLU ASN PRO VAL ARGTYR GLY GLN CYS TRP VAL PHE ALA  CTA TTG GAA TAC CGG AGC TCT GAG AAT CCAGTC CGG TAT GGC CAA TGC TGG GTT TTT GCT  1042  GLY VAL PHE ASN THR PHELEU ARG CYS LEU GLY ILE PRO ALA ARG ILE VAL THR ASN TYR  GGT GTC TTT AACACA TTT TTA CGA TGC CTT GGA ATA CCA GCA AGA ATT GTT ACC AAT TAT  1102 PHE SER ALA HIS ASP ASN ASP ALA ASN LEU GLN MET ASP ILE PHE LEU GLU GLUASP GLY  TTC TCT GCC CAT GAT AAT GAT GCC AAT TTG CAA ATG GAC ATC TTC CTGGAA GAA GAT GGG  1162  ASN VAL ASN SER LYS LEU THR LYS ASP SER VAL TRPASN TYR HIS CYS TRP ASN GLU ALA  AAC GTG AAT TCC AAA CTC ACC AAG GAT TCAGTG TGG AAC TAC CAC TGC TGG AAT GAA GCA  1222  TRP MET THR ARG PRO ASPLEU PRO VAL GLY PHE GLY GLY TRP GLN ALA VAL ASP SER THR  TGG ATG ACA AGGCCT GAC CTT CCT GTT GGA TTT GGA GGC TGG CAA GCT GTG GAC AGC ACC  1282 PRO GLN GLU ASN SER ASP GLY MET TYR ARG CYS GLY PRO ALA SER VAL GLN ALAILE LYS  CCC CAG GAA AAT AGC GAT GGC ATG TAT CGG TGT GGC CCC GCC TCG GTTCAA GCC ATC AAG  1342  HIS GLY HIS VAL CYS PHE GLN PHE ASP ALA PRO PHEVAL PHE ALA GLU VAL ASN SER ASP  CAC GGC CAT GTC TGC TTC CAA TTT GAT GCACCT TTT GTT TTT GCA GAG GTC AAC AGC GAC  1402  LEU ILE TYR ILE THR ALALYS LYS ASP GLY THR HIS VAL VAL GLU ASN VAL ASP ALA THR  CTC ATT TAC ATTACA GCT AAG AAA GAT GGC ACT CAT GTG GTG GAA AAT GTG GAT GCC ACC  1462 HIS ILE GLY LYS LEU ILE VAL THR LYS GLN ILE GLY GLY ASP GLY MET MET ASPILE THR  CAC ATT GGG AAA TTA ATT GTG ACC AAA CAA ATT GGA GGA GATGGC|ATG ATG GAT ATT ACT  1522  ASP THR TYR LYS PHE GLN GLU GLY GLN GLUGLU GLU ARG LEU ALA LEU GLU THR ALA LEU  GAT ACT TAC AAA TTC CAA GAA GGTCAA GAA GAA GAG AGA TTG GCC CTA GAA ACT GCC CTG  20 mer|  1582  MET TYRGLY ALA LYS LYS PRO LEU ASN THR GLU GLY VAL MET LYS SER ARG SER ASN VAL ATG TAC GGA GCT AAA AAG CCC CTC AAC ACA GAA GGT GTC ATG AAA TCA AGG TCCAAC GTT  1642  ASP MET ASP PHE GLU VAL GLU ASN ALA VAL LEU GLY LYS ASPPHE LYS LEU SER ILE THR  GAC ATG GAC TTT GAA GTG GAA AAT GCT GTG CTG GGAAAA GAC TTC AAG CTC TCC ATC ACC  1702  PHE ARG ASN ASN SER HIS ASN ARGTYR THR ILE THR ALA TYR LEU SER ALA ASN ILE THR  TTC CGG AAC AAC AGC CACAAC CGT TAC ACC ATC ACA GCT TAT CTC TCA GCC AAC ATC ACC  1762  PHE TYRTHR GLY VAL PRO LYS ALA GLU PHE LYS LYS GLU THR PHE ASP VAL THR LEU GLU TTC TAC ACC GGG GTC CCG AAG GCA GAG TTC AAG AAG GAG ACG TTC GAC GTG ACGCTG GAG  1822  PRO LEU SER PHE LYS LYS GLU ALA VAL LEU ILE GLN ALA GLYGLU TYR MET GLY GLN LEU  CCC TTG TCC TTC AAG AAA GAG GCG GTG CTG ATC CAAGCC GGC GAG TAC ATG GGT CAG CTG  1882  LEU GLU GLN ALA SER LEU HIS PHEPHE VAL THR ALA ARG ILE ASN GLU THR ARG ASP VAL  CTG GAA CAA GCG TCC CTGCAC TTC TTT GTC ACA GCT CGC ATC AAT GAG ACC AGG GAT GTT  1942  LEU ALALYS GLN LYS SER THR VAL LEU THR ILE PRO GLU ILE ILE ILE LYS VAL ARG GLY CTG GCC AAG CAA AAG TCC ACC GTG CTA ACC ATC CCT GAG ATC ATC ATC AAG GTCCGT GGC  2002  THR GLN VAL VAL GLY SER ASP MET THR VAL THR VAL GLN PHETHR ASN PRO LEU LYS GLU  ACT CAG GTA GTT GGT TCT GAC ATG ACT GTG ACA GTTCAG TTT ACC AAT CCT TTA AAA GAA  2062  THR LEU ARG ASN VAL TRP VAL HISLEU ASP GLY PRO GLY VAL THR ARG PRO MET LYS LYS  ACC CTG CGA AAT GTC TGGGTA CAC CTG GAT GGT CCT GGA GTA ACA AGA CCA ATG AAG AAG  2122  MET PHEARG GLU ILE ARG PRO ASN SER THR VAL GLN TRP GLU GLU VAL CYS ARG PRO TRP ATG TTC CGT GAA ATC CGG CCC AAC TCC ACC GTG CAG TGG GAA GAA GTG TGC CGGCCC TGG  2182  VAL SER GLY HIS ARG LYS LEU ILE ALA SER MET SER SER ASPSER LEU ARG HIS VAL TYR  GTC TCT GGG CAT CGG AAG CTG ATA GCC AGC ATG AGCAGT GAC TCC CTG AGA CAT GTG TAT  2242  GLY GLU LEU ASP VAL GLN ILE GLNARG ARG PRO SER MET SSS ATGCACAGGAAGCTGAGATGAAC  GGC GAG CTG GAC GTG CAGATT CAA AGA CGA CCT TCC ATG TGA  2307CCTGGCATTTGGCCTCTTGTAGTCTTGGCTAAGGAAATTCTAACGCAAAAATAGCTCTTGCTTTGACTTAGGTGTGAAGA 2387CCCAGACAGGACTGCAGAGGGCCCCAGAGTGGAGATCCCACATATTTCAAAAACATACTTTTCCAAACCCAGGCTATTCG 2467GCAAGGAAGTTAGTTTTTAATCTCTCCACCTTCCAAAGAGTGCTAAGCATTAGCTTTAATTAAGCTCTCATAGCTCATAA 2547GAGTAACAGTCATCATTTATCATCACAAATGGCTACATCTCCAAATATCAGTGGGCTCTCTTACCAGGGAGATTTGCTCA 2627ATACCTGGCCTCATTTAAAACAAGACTTCAGATTCCCCACTCAGCCTTTTGGGAATAATAGCACATGATTTGGGCTCTAG 2707AATTCCAGTCCCCTTTCTCGGGGTCAGGTTCTACCCTCCATGTGAGAATATTTTTCCCAGGACTAGAGCACAACATAATT 2787TTTATTTTTGGCAAAGCCAGAAAAAGATCTTTCATTTTGCACCTGCAGCCAAGCAAATGCCTGCCAAATTTTAGATTTAC 2867CTTGTTAGAAGAGGTGGCCCCATATTAACAAATTGCATTTGTGGGAAACTTAACCACCTACAAGGAGATAAGAAAGCAGG 2947TGCAACACTCAAGTCTATTGAATAATGTAGTTTTGTGATGCATTTTATAGAATGTGTCACACTGTGGCCTGATCAGCAGG 3027AGCCAATATCCCTTACTTTAACCCTTTCTGGGATGCAATACTAGGAAGTAAAGTGAAGAATTTATCTCTTTAGTTAGTGA 3107TTATATTTCACCCATCTCTCAGGAATCATCTCCTTTGCAGAATGATGCAGGTTCAGGTCCCCTTTCAGAGATATAATAAG 3187CCCAACAAGTTGAAGAAGCTGGCGGATCTAGTGACCAGATATATAGAAGGACTGCAGCCACTGATTCTCTCTTGTCCTTC 3267ACATCACCATTTTGAGACCTCAGCTTGGCACTCAGGTGCTGAAGGGTAATATGGACTCAGCCTTGCAAATAGCCAGTGCT 3347AGTTCTGACCCAACCACAGAGGATGCTGACATCATTTGTATTATGTTCCAAGGCTACTACAGAGAAGGCTGCCTGCTATG 3427TATTTGCAAGGCTGATTTATGGTCAGAATTTCCCTCTGATATGTCTAGGGTGTGATTTAGGTCAGTAGACTGTGATTCTT 3507AGCAAAAAATGAACAGTGATAAGTATACTGGGGGCAAAATCAGAATGGAATGCTCTGGTCTATATAACCACATTTCTGAG 3587CCTTTGAGACTGTTCCTGAGCCTTCAGCACTAACCTATGAGGGTGAGCTGGTCCCCTCTATATATACATCATACTTAACT 3667TTACTAAGTAATCTCACAGCATTTGCCAAGTCTCCCAATATCCAATTTTAAAATGAAATGCATTTTGCTAGACAGTTAAA 3747CTGGCTTAACTTAGTATATTATTATTAATTACAATGTAATAGAAGCTTAAAATAAAGTTAAACTGATTATAAAAAAAAAA 3827AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TABLE 4 p FXIII-13

p FXIII-C4

pMB259

aa = amino acidsAppendixExample 1 from German Patent Application P 36 24 453.8a) Construction of an expression vector for animal cells

The plasmid pSV2dhfr (Lee et al., loc. cit.) was cut with HindIII andEcoRI, and the 2.65 kb vector fragment which has the SV40 early promotorwas isolated. A 67 bp HindIII-EcoRI fragment from pUC12 STOP (Broker andAmann, Appl. Microbiol. Biotechnol. 23 (1986) 294-296) was ligated intothe vector which had been pretreated in this way, which results in theplasmid pSV2 STOP. On the 67 bp fragment from pUC12 STOP there aretranslation stop codons in all three reading frames. pSV2 STOP waslinearized with SacI, and the resulting 3′ protruding end was removedusing the 3′→5′ exonuclease activity of DNA polymerase I. Then digestionwith EcoRI was carried out. After ligation with an EcoRI-HpaI fragment133 bp in size from pBB3 (B. Bourachot et al., EMBO J. 1 (1982)895-900), which has the SV40 polyadenylation signal for earlytranscripts, it was possible to obtain the expression vector pSVA STOP1.

The polyadenylation site can also be isolated from the vector pIG6(Bourachot et al., loc. cit.). It is possible in exactly the same way toisolate from the SV40 gene the 133 bp BamHI-HapI fragment, to fill inthe BamHI cleavage site, and to attach an EcoRI linker.

pSVA STOP1 thus has, between the SV40 early promotor and the SV40polyadenylation signal for early transcripts, a cloning polylinker withthree unique restriction sites (HindIII-SalI-XbaI) and a sequence withtranslation stops in all three reading frames.

c) Construction of DHFR expression vectors for co-transfection

The starting point for the DHFR vectors used for the co-transfection wasthe plasmid pMTVdhfr (Lee et al., loc. cit.). pMTVdhfr was cut withBglII, and the protruding 5′ ends of the DNA were filled in using DNApolymerase I (Klenow fragment). After digestion with EcoRI, a fragment4.47 kb in size was isolated and ligated with a 133 bp EcoRI-HpaIfragment from pBB3 (Bourachot et al., loc. cit.). The new plasmidpMTVAdhfr has the mouse DHFR cDNA flanked by MMTV-LTR and the SV40polyadenylation site for early transcripts.

pSVOAdhfr was obtained from pMTVAdhfr by deletion of a HindIII fragmentwhich is 1450 bp in size and has the MMTV-LTR.

Neither pMTVAdhfr nor pSVOAdhfr have mRNA splice sites.

1. A method for producing factor XIIIa comprising operably inserting aDNA comprising the sequence set forth in FIG. 6, or naturally occurringalleles thereof, into a suitable vector, transforming an appropriatehost cell with said vector, and expressing Factor XIIIa.
 2. A method forproducing a variant of a Factor XIIIa comprising operably inserting aDNA comprising a sequence from nucleotide position 82 to nucleotideposition 2280 of FIG. 6, or degenerate variants thereof, into a suitableexpression vector, transforming an appropriate host cell with saidvector, and expressing said variant of a Factor XIIIa, wherein saidvariant of a Factor XIIIa has fibrin fusion catalysis activity.
 3. Amethod for producing a variant of a Factor XIIIa comprising operablyinserting a fragment of a DNA comprising the sequence set forth in FIG.6, or naturally occurring alleles thereof, into a suitable expressionvector, transforming an appropriate host cell with said vector, andexpressing said variant of a Factor XIIIa, wherein said variant of aFactor XIIIa has fibrin fusion catalysis activity.
 4. A method forproducing a variant of a Factor XIIIa comprising operably inserting aDNA comprising a sequence from nucleotide position 199 to nucleotideposition 2280 of FIG. 6, or degenerate variants thereof, into a suitableexpression vector, transforming an appropriate host cell with saidvector, and expressing said variant of a Factor XIIIa, wherein saidvariant of a Factor XIIIa has fibrin fusion catalysis activity.
 5. Themethod according to claim 1, wherein the DNA comprises the sequence setforth in FIG.
 6. 6. The method according to claim 3, wherein the DNAcomprises the sequence set forth in FIG.
 6. 7. The method according toclaim 1, 2, 3 or 4, wherein the vector further comprises the strong ADHIpromoter.
 8. The method according to claim 1, 2, 3 or 4, wherein thehost cell is an animal cell.
 9. The method according to claim 8, whereinthe animal cell is a CHO cell.
 10. The method according to claim 1, 2, 3or 4, wherein the host cell is an Escherichia coli cell.
 11. The methodaccording to claim 1, 2, 3 or 4, wherein the host cell is a yeast cell.12. The method according to claim 11, wherein the yeast cell is aSaccharomyces cerevisiae cell.
 13. The method according to claim 7,wherein the host cell is a yeast cell.
 14. The method according to claim13, wherein the yeast cell is a Saccharomyces cerevisiae cell.
 15. Themethod according to claim 5 or 6, wherein the vector further comprisesthe strong ADHI promoter.
 16. The method according to claim 5 or 6,wherein the host cell is an animal cell.
 17. The method according toclaim 16, wherein the animal cell is a CHO cell.
 18. The methodaccording to claim 5 or 6, wherein the host cell is an Escherichia colicell.
 19. The method according to claim 5 or 6, wherein the host cell isa yeast cell.
 20. The method according to claim 19, wherein the yeastcell is a Saccharomyces cerevisiae cell.
 21. The method according toclaim 15, wherein the host cell is a yeast cell.
 22. The methodaccording to claim 21, wherein the yeast cell is a Saccharomycescerevisiae cell.
 23. An isolated DNA comprising the DNA sequence setforth in FIG. 6 or naturally occurring alleles thereof.
 24. An isolatedDNA comprising the DNA sequence from nucleotide position 82 tonucleotide position 2280 of FIG. 6 or degenerate variants thereof. 25.An isolated DNA comprising a fragment of the DNA sequence set forth inFIG. 6 or naturally occurring alleles thereof.
 26. An isolated DNAcomprising the DNA sequence from nucleotide position 199 to nucleotideposition 2280 of FIG. 6 or degenerate variants thereof.
 27. A vectorcomprising the isolated DNA according to claims 23, 24, 25, or
 26. 28. Avector according to claim 27, further comprising the strong ADHIpromoter.
 29. A host cell comprising the vector according to claim 27.30. The host cell according to claim 29, wherein the host cell is ananimal cell.
 31. The host cell according to claim 30, wherein the animalcell is a CHO cell.
 32. The host cell according to claim 29, wherein thehost cell is an Escherichia coli cell.
 33. The host cell according toclaim 29, wherein the host cell is a yeast cell.
 34. The host cellaccording to claim 33, wherein the yeast cell is a Saccharomycescerevisiae cell.
 35. The host cell comprising the vector according toclaim
 28. 36. The host cell according to claim 35, wherein the host cellis a yeast cell.
 37. The host cell according to claim 36, wherein theyeast cell is a Saccharomyces cerevisiae cell.